Compositions and methods for enzymatic detachment of bacterial and fungal biofilms

ABSTRACT

Isolated nucleic acid sequences and amino acid sequences for soluble, β-N-acetylglucosaminidase or active fragments or variants thereof which promote detachment of bacterial cells from a biofilm are provided. An isolated mutant bacteria which forms biofilm colonies which tightly adhere to surface but which are unable to release cells into the medium or spread over the surface is also provided. In additions, methods are described for modulating detachment of bacterial cells from biofilm by mutating soluble,β-N-acetylglucosamimidase or altering its expression or activity are also provided. Also provided are compositions, methods and devices for preventing, inhibiting and treating bacterial infections.

This application claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 60/435,817, filed Dec. 20, 2002, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides isolated nucleic acid sequences and aminoacid sequences encoded thereby for the protein, soluble,β-N-acetylglucosaminidase or dispersin B, and active fragments andvariants thereof, which promote detachment of bacterial cells frombiofilms. Vectors comprising the nucleic acid sequences as well as hostcells expressing the dispersin B protein or active fragments or variantsthereof are also provided. A biofilm detachment mutant of A.actinomycetemcomitans is also described. The nucleic acid and amino acidsequences of the present invention are useful in methods for modulatingdetachment of bacterial or fungal cells from biofilms as well as inmethods for identifying agents which modulate detachment of bacterial orfungal cells from biofilms. Thus, these nucleic acid and amino acidsequences and agents are expected to be useful in the prevention andtreatment of bacterial or fungal infections and in disinfectant andantiseptic solutions.

BACKGROUND OF THE INVENTION

Biofilms are populations of bacteria or fungi growing attached to aninert or living surface. Mounting evidence has shown that biofilmsconstitute a significant threat to human health. The Public HealthService estimates that biofilms are responsible for more than 80% ofbacterial infections in humans (National Institutes of Health, 1998 RFA#DE-98-006). Examples of diseases caused by biofilms include dentalcaries, periodontitis, cystic fibrosis pneumonia, native valveendocarditis, and otitis media (Costerton et al. Science 1999284:1318-1322), as well as infection of various medical devices such asurinary catheters, mechanical heart valves, cardiac pacemakers,prosthetic joints, and contact lenses (Donlan, R. M. 2001 EmergingInfect. Dis. 7:277-281). Fungi also form biofilms of clinicalsignificance, for example Candida infections. Biofilm infections afflicttens of millions of patients in the U.S. annually and require asignificant expenditure of health care dollars (Costerton et al. Science1999 284:1318-1322). Bacteria growing in biofilms exhibit increasedresistance to antimicrobial agents and are nearly impossible toeradicate. New methods for treating biofilm infections are needed.

Bacteria in a biofilm are enmeshed in an extracellular polysaccharide(EPS) substance that holds the bacteria together in a mass, and firmlyattaches the bacterial mass to the underlying surface. Previous studieshave demonstrated that enzymes that degrade EPS are capable of causingthe detachment of cells from biofilms. For example, over expression ofalginate lyase, an enzyme that catalyzes the degradation of the EPSalginate, causes colonies of Pseudomonas aeruginosa to become lessadherent to surfaces (Boyd, A. and Chakrabarty, A. M. Appl. Environ.Microbiol. 1994 60:2355-2359). Alginate lyase has been suggested for usein treating P. aeruginosa infections in the lungs of cystic fibrosispatients (Mrsny et al. Pulm. Pharmacol. 1994 7:357-366). A similarpolysaccharide lyase has been shown to be produced by P. fluorescens(Allison et al. FEMS Microbiol. Lett. 1998 167:179-184). Two otherEPS-degrading enzymes, endo-β-1,4-mannanase from the plant pathogenXanthomonas campestris (Dow et al. Proc. Nat. Acad. Sci. USA 2003100:10995-11000) and disaggretase from the methanogenic archaebacteriumMethanosarcina mazei (Liu et al. Appl. Environ. Microbiol. 198549:608-613), have also been shown to cause biofilm cell detachment. Inthe case of X. campestris, production of the EPS-degrading enzyme wasrequired for full virulence of the bacteria in plants. Detachment ofcells from biofilm colonies of the dental pathogen Streptococcus mutanswas shown to be caused by an unidentified endogenous enzymatic activity(Lee et al. Infect. Immun. 1996 64:1035-1038). A complex mixture ofpolysaccharide-hydrolyzing enzymes was shown to remove biofilms fromsteel and polypropylene substrata (Johansen et al. Appl. Environ.Microbiol. 1997 63:3724-3728). These findings indicate thatEPS-degrading enzymes can potentially be used as agents to remove.biofilms from surfaces.

Although enzymes are commonly used to remove biofilms in industrialenvironments, no studies have investigated the potential use of enzymesas agents for the removal of biofilms in clinical environments. Ofparticular concern in the clinic are biofilm infections of indwellingmedical devices, especially intravascular catheters. Catheter infectionsare common in hospitalized patients and are associated with high levelsof morbidity and mortality. A promising new approach to treating theseinfections is the use of catheters that are coated or impregnated withantimicrobial agents such as antibiotics (Schierholz et al. J.Antimicrobial. Chemother. 2000 46:45-50), silver (Bechert et al.Infection 1999 27:S24-S29), and peptide quorum-sensing inhibitors(Balaban et al. J. Infect. Dis. 2003 187:625-630). Numerous studies havedemonstrated that medical devices with antimicrobial activity decreasethe risk of bacterial colonization and infection (Tcholakian, R. K. andRaad, I. I. Antimicrob. Agents Chemother. 2001 45:1990-1993).

The present invention provides isolated proteins and active fragmentsand variants thereof and nucleic acid sequences encoding such proteinsand active fragments and variants thereof involved in detachment ofbacterial cells. Methods for modulating detachment of biofilm cells ofbacteria or fungi and identifying agents which modulate bacterial orfungal detachment via these proteins and active fragments and variantsthereof and/or nucleic acid sequences are also provided.

SUMMARY OF THE INVENTION

An object of the present invention is to provide isolated proteins andactive fragments and variants thereof which promote detachment ofbacterial or fungal cells from a biofilm. The isolated proteins arereferred to herein as soluble, β-N-acetylglucosaminidase or dispersin B.

Another object of the present invention is to provide isolated nucleicacid sequences encoding soluble, β-N-acetylglucosaminidase and activefragments and variants thereof as well as vectors comprising thesesequences and host cells expressing the vectors.

Another object of the present invention is to provide methods formodulating detachment of bacterial or fungal cells from biofilms. In oneembodiment the method comprises mutating the bacterial cells to inhibitdetachment of bacterial cells from biofilms. In another embodiment, themethod comprises increasing expression and/or levels of soluble,β-N-acetylglucosaminidase or active fragments or variants thereof in thebacterial or fungal cells so that detachment is increased. In yetanother embodiment, the method comprises decreasing expression and/orlevels of soluble, β-N-acetylglucosaminidase or active fragments orvariants thereof or inhibiting activity of soluble,β-N-acetylglucosaminidase or active fragments or variants thereof sothat detachment of bacterial cells is decreased.

Another object of the present invention is to provide an isolated mutantof Actinobacillus actinomycetemcomi tans which forms biofilm colonieswhich tightly adhere to surface but which are unable to release cellsinto the medium or spread over the surface.

Another object of the present invention is to provide a method foridentifying agents which modulate detachment of bacterial or fungalcells from biofilms which comprises assessing the ability of an agent tomodulate activity and/or levels and/or expression of soluble,β-N-acetylglucosaminidase.

Another object of the present invention is to provide compositions andmethods for using these compositions to prevent the dissemination ofinfectious bacteria via administration of an agent which inhibitssoluble, β-N-acetylglucosaminidase expression and/or activity in thebacterial cells.

Another object of the present invention is to provide compositions andmethods for preventing or inhibiting attachment of infectious bacteriaor fungi to a surface or removing infectious bacteria or fungi from asurface which comprises treating the surface with soluble,β-N-acetylglucosaminidase, or an active fragment or variant thereof.

Yet another object of the present invention is to provide PCR primerpairs and kits comprising such primer pairs that can be used to identifyadditional bacterial species with homologues of soluble,β-N-acetylglucosaminidase.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 provides a clustal alignment of exemplary dispersin B orthologsof the present invention from A. actinomycetemcomitans strain CU1000N(SEQ ID NO:2), A. actinomycetemcomitans strain IDH781 (SEQ ID NO:6),Haemophilus aphrophilus strain NJ8700 (SEQ ID NO:8), A. ligniersiistrain 19393 (SEQ ID NO:4), and A. pleuropneumoniae strain IA5 (SEQ IDNO:10).

DETAILED DESCRIPTION OF THE INVENTION

The small, gram-negative coccobacillus Actinobacillusactinomycetemcomitans is a common inhabitant of the human oral cavity(King, E. O. and Tatum, H. W. J. Infect. Dis. 1962 111:85-94). A.actinomycetemcomitans has been implicated as the causative agent oflocalized juvenile periodontitis, a severe and rapid form of periodontaldisease that affects adolescents (Zambon, J. J. J. Clin. Periodontol1985 12:1-20). A. actinomycetemcomitans can also enter the submucosa andcause infective endocarditis and other non-oral infections (Kaplan etal. Rev. Infect. Dis. 1989 11:46-63).

When cultured in broth, fresh clinical isolates of A.actinomycetemcomitans form tenacious biofilms on surfaces such as glass,plastic and saliva-coated hydroxyapatite (Fine et al. Arch. Oral. Biol.1999 44:1063-1076; Fine et al. Microbiol. 1999 145:1335-1347; Fine etal. Arch. Oral Biol. 2001 46:1065-1078; Haase et al. Infect. Immun. 199967:2901-2908; Inouye et al. FEMS Microbiol. Lett. 1990 69:13-18;Kachlany et al. J. Bacteriol. 2000 182:6169-6176; Kachlany et al. Mol.Microbiol. 2001 40:542-554; Kagermeier, A. S., and London, J. Infect.Immun. 1985 47:654-658; Kaplan, J. B., and Fine, D. H. Appl. Environ.Microbiol. 2002 68:4943-4950; King, E. O. and Tatum, H. W. J. Infect.Dis. 1962 111:85-94; Rosan et al. Oral. Microbiol. Immunol. 19883:58-63). Nearly all of the cells grow attached to the surface while thebroth remains clear and is often sterile (Fine et al. Arch. Oral. Biol.1999 44:1063-1076). The dense biofilm that forms on the surface isresistant to removal by agents such as detergents, proteases, heat,sonication and vortex agitation (Fine et al. Arch. Oral. Biol. 199944:1063-1076), and can be removed only by mechanical scraping. A.actinomycetemcomi tans biofilm colonies exhibit increased resistance toantimicrobial agents when compared to cells grown in planktonic form(Fine et al. J. Clin. Periodontol. 2001 28:697-700).

Tight adherence has been shown to play an important role in the abilityof A. actinomycetemcomitans to colonize the mouths of rats (Fine et al.Arch. Oral Biol. 2001 46:1065-1078.), and is believed to have an equallyimportant role in its ability to colonize humans. The tight adherence tosurfaces is dependent on the presence of long, bundled pili (fimbriae)that form on the surface of the cell (Inouye et al. FEMS Microbiol.Lett. 1990 69:13-18; Rosan et al. Oral. Microbiol. Immunol. 19883:58-63). Mutations in flp-1, which encodes the major pilin proteinsubunit, result in cells that fail to produce fimbriae or adhere tosurfaces (Kachlany et al. Mol. Microbiol. 2001 40:542-554).

Biofilm colonies of A. actinomycetemcomitans have been shown to releasecells into liquid medium which then attach to the surface of the culturevessel and form new colonies, enabling the biofilm to spread (Kaplan, J.B. and Fine D. H. Appl. Environ. Microbiol. 2002 68: 4943-4950.).

One aspect of the present invention relates to a mutant of A.actinomycetemcomitans that forms biofilm colonies which are tightlyadherent to surfaces but which are unable to release cells into themedium or spread over the surface. The biofilm detachment mutant of A.actinomycetemcomitans is referred to herein as mutant JK1023. To producethe A. actinomycetemcomitans biofilm detachment mutant JK1023, the A.actinomycetemcomitans strain CU1000N was mutagenized with transposonIS903φkan. The mutant strain (designated JK1023) was then isolated. Thismutant strain displays a colony morphology on agar that is rougher thanthe wild-type A. actinomycetemcomitans rough-colony phenotype (Fine etal. Microbiol. 1999 145:1335-1347; Haase et al. Infect. Immun. 199967:2901-2908; Inouye et al. FEMS Microbiol. Lett. 1990 69:13-18). JK1023colonies had a hard texture and were extremely difficult to remove fromthe agar surface. When cultured in broth, strain JK1023 produced biofilmcolonies which were similar in size and shape to those of the wild-typestrain, but which failed to produce satellite colonies on the surface ofthe culture vessel. Adherence of JK1023 cells to polystyrene was equalto that of wild-type strain CU1000N as measured by a 96-well microtiterplate binding assay.

To demonstrate that biofilm colonies of mutant strain JK1023 of thepresent invention were deficient in biofilm cell detachment, biofilmcolonies were grown for 24 hours on polystyrene rods suspended in brothin the wells of a 24-well microtiter plate. The amount of biofilm celldetachment was then quantified by staining the bacteria growing on thebottom of the well with crystal violet. Colonization at the bottom ofthe well results from cells that detach from the biofilm coloniesgrowing on the polystyrene rod and fall to the bottom of the well. Inthis assay, biofilm colonies of strain JK1023 produced significantlyless growth on the bottom of the well than the wild-type strain (P<0.01,unpaired two-tailed t test). These data indicate that mutant strainJK1023 exhibited a wild-type surface attachment phenotype but adecreased biofilm cell detachment phenotype when compared to thewild-type strain CU1000N.

DNA sequence analysis of the region surrounding the transposon insertionsite of this mutant strain revealed the insertion to be in a 1,143 bpopen reading frame designated herein as dspB. The dspB gene from strainCU1000 was predicted to encode a protein, referred to herein asdispersin B, having 381 amino acid residues with a molecular mass of43.3 kDa. The 5′ end of dspB contained a predicted signal peptide,suggesting that dispersin B may be a secreted protein.

In addition to A. actinomycetemcomitans, dspb nucleic acid sequences orfragments have also been isolated from Actinobacillus pleuropneumonaie,Haemophilus aphrophilus and Actinobacillus ligniersii. DspB is notpresent in the genomes of Haemophilus influenzae, Pasteurella multicido,Mannheimia haemolytica, Actinobacillus equuli and Haemophilus ducreyiamong the strains that were tested.

Accordingly, another aspect of the present invention relates to nucleicacid sequences encoding dispersin B or active fragments and variantsthereof as well as amino acid sequences of dispersin B and activefragments and variants thereof. Also encompassed by the presentinvention are vectors comprising these nucleic acid sequences as well ashost cells comprising the vectors which express dispersin B or an activefragment thereof.

By the term “nucleic acid sequence” as used herein it is meant toinclude, but is not limited to, unmodified RNA or DNA or modified RNA orDNA. Thus, by nucleic acid sequence it is meant to be inclusive ofsingle- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescontaining DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.Further, the DNA or RNA sequences of the present invention may comprisea modified backbone and/or modified bases. A variety of modifications toDNA and RNA are known in the art for multiple useful purposes. The term“nucleic acid sequence” as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms of nucleicacid sequences, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells.

The DNA sequence of dspB from strain CU1000 was deposited into GenBankunder accession no. AY228551 and released on Aug. 4, 2003. The nucleicacid sequence for this DNA is SEQ ID NO:1. Nucleic acid sequencesencoding orthologs of dispersin B protein have been identified in A.ligniersii strain 19393, A. actinomycetemcomitans strain IDH781,Haemophilus aphrophilus strain NJ8700 and A. pleuropneumoniae strain IA5and are depicted in SEQ ID NO:3, 5, 7 and 9, respectively. Accordingly,preferred isolated nucleic acid sequences of the present inventioncomprise SEQ ID NO:1, 3, 5, 7 or 9.

Also included within the present invention are allelic variants of theexemplified dspB nucleic acid sequences of SEQ ID NO:1, 3, 5 7 or 9encoding proteins with similar enzymatic activities to dispersin B andnucleic acid sequences with substantial percent sequence identity to theexemplified dspB nucleic acid sequences of SEQ ID NO: 1, 3, 5, 7 or 9encoding proteins with similar enzymatic activities.

By the term “allelic variant” as used herein it is meant one of two ormore alternative naturally occurring forms of a gene, each of whichcomprises a unique nucleic acid sequence. Allelic variants encompassedby the present invention encode proteins with similar or identicalenzymatic activities.

The term “percent sequence identity” as used herein with respect tonucleic acid sequences refers to the residues in two nucleic acidsequences which are the same when aligned for maximum correspondence.The length of sequence identity comparison is preferably over a lengthof at least about 9 contiguous nucleotides, more preferably about 18contiguous nucleotides, and even more preferably at least about 30 to 50contiguous nucleotides or more. Various algorithms well known in the artare available for measuring nucleic acid sequence identity. Examplesinclude, but are not limited to, FASTA (including FASTA2 and FASTA3),Gap and Bestfit, which are programs in Wisconsin Package Version 10.0,Genetics Computer Group (GCG), Madison, Wis.

By “substantial percent sequence identity” when referring to a nucleicacid sequence or fragment thereof, of the present invention, it is meantthat when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), atleast about 50% of the nucleotide bases as measured by any well knownalgorithm of sequence identity, such as FASTA, BLAST or Gap are thesame. For purposes of the present invention, more preferably, at leastabout 60% to 70%, even more preferably 80% to 90%, and most preferablyat least about 95-98% of the nucleotide bases, as measured by any wellknown algorithm of sequence identity, such as FASTA, BLAST or Gap, areidentical.

Nucleic acid sequences sharing substantial percent sequence identity andencoding proteins with similar functional activity are referred toherein as orthologues.

Deduced amino acid sequences of dispersin B and exemplary orthologuesthereof are shown in FIG. 1. Specifically, the amino acid sequence ofdispersin B of A. actinomycetemcomitans strain CU1000N (SEQ ID NO:2),and orthologs of dispersin B from A. actinomycetemcomitans strain IDH781(SEQ ID NO:6), Haemophilus aphrophilus strain NJ8700 (SEQ ID NO:8), A.ligniersii strain 19393 (SEQ ID NO:4), and A. pleuropneumoniae strainIA5 (SEQ ID NO:10) are shown.

There are similarities between the amino acid sequence of dispersin Band these orthologs and the consensus sequence of the family 20 glycosylhydrolase. More specifically, amino acid residues 40 to 297 of thepredicted dispersin B protein sequence are homologous to the catalyticdomain of the family 20 glycosyl hydrolases (NCBI Conserved DomainDatabase accession Number pfam00728). This family of enzymes includesbacterial chitinases, chitobiases and lacto-N-biosidases (Sano et al. J.Biol. Chem. 1993 268:18560-18566; Tews et al. Gene 1996 170:63-67;Tsujibo et al. Biochim. Biophys. Acta 1998 1425:437-440.), andeukaryotic hexosaminidases (Graham et al. J. Biol. Chem. 1988263:16823-16829). A protein related to A. actinomycetemcomitansdispersin B is lacto-N-biosidase of Lactococcus lactis (GenBankaccession no. AAK05592), which displays 28% identity over 281 amino acidresidues not counting gaps and terminal extensions. Similarity betweendispersin B and lacto-N-biosidases is high in the regions surroundingArg47 and the acidic amino acid pair Asp202 and Glu203. These residueshave been shown to participate in substrate binding and catalysis inother family 20 glycosyl hydrolases (Mark et al. J. Biol. Chem. 2001276:10330-10337; Mark et al. J. Biol. Chem. 1998 273:19618-19624; Praget al. J. Mol. Biol. 2000 300:611-617). The C-terminal half of dispersinB contained three Trp residues that were conserved in L. lactislacto-N-biosidase (positions 236, 279, and 353). Multiple Trp residuesare present in the C-terminal regions of the catalytic domains of allfamily 20 glycosyl hydrolases (Graham et al. J. Biol. Chem. 1988263:16823-16829; Tews et al. Gene 1996 170:63-67). These Trp residuesline the part of the substrate binding pocket that is complementary tothe hydrophobic surfaces of the hexosamine sugar ring (Tews et al.Nature Struct. Biol. 1996 3:638-648). It is expected that mutation ofamino acids in these regions of dispersin B and its orthologs will alterenzymatic activity.

In a preferred embodiment an isolated amino acid sequence of the presentinvention comprises SEQ ID NO:2, 4, 6, 8 or 10 or an active fragment orvariants thereof. Preferred active fragments are those comprising aportion of the amino acid sequence of SEQ ID NO:2, 4, 6, 8 or 10 withsimilarities to the consensus sequence of the family 20 glycosylhydrolase.

“Active variants” or “functionally equivalent variants” as used hereinare polypeptide sequences structurally different from the dispersin Bprotein, but having no significant functional difference from theprotein. For example, when orthologous polypeptide sequences fromvarious strains of A. actinomycetemcomitans are aligned, divergence inamino acid sequence is observed, usually 0 to 10 percent (Kaplan et al.Oral Microbiol. Immunol. December 2002 17:354-359; Kaplan et al. Infect.Immun. 2001 69:5375-5384). Proteins displaying this amount of divergenceare considered functionally equivalent variants because of the fact thatmixing of genetic alleles that encode these variants is often observedin populations (Kaplan et al. Oral Microbol. Immunol. December 200217:354-359). The dispersin B sequence from A. actinomycetemcomitansstrain IDH781 (SEQ ID NO:6), therefore, is expected to be a functionallyequivalent or active variant of SEQ ID NO:2, and is included in thescope of the present invention. Similarly, dispersin B sequences fromother strains of A. actinomycetemcomitans, such as those that exhibitdifferent serotypes, restriction fragment length polymorphism genotypes,16S ribosomal RNA genotypes, or arbitrarily-primed PCR genotypes thatare commonly observed among phylogenetically diverse strains isolatedfrom different subjects (Kaplan et al. J. Clin. Microbiol. 200240:1181-1187; Kaplan et al., Oral Microbiol. Immunol. December 200217:354-359), are also expected to be functionally equivalent or activevariants of SEQ ID NO:2, and are included in the scope of the presentinvention.

Similarly, orthologous proteins from phylogenetically diverse species ofbacteria are usually functionally equivalent or active variants, asevidenced by the fact that a common method for cloning genes of interestinto plasmids is to screen a plasmid library for plasmids thatcomplement a genetic mutation in a different species of bacteria (Kaplanet al. J. Mol. Biol. 1985 183:327-340). This is especially true ofbacterial enzymes. Orthologous enzymes of different bacterial speciescan exhibit up to 50% divergence or greater, yet still utilize theidentical substrate, catalyze the same chemical reaction, and producethe same product. This sequence divergence results from genetic driftcoupled with fixation of selected genetic changes in the population. Thegenetic changes that are selected and fixed are those that altercharacteristics of the enzyme other than substrate, reaction, andproduct, as for example, reaction rate, pH optimum, temperature optimum,level of expression, and interactions with other enzymes, such thatthese genetic changes confer upon a bacterial cell a selective advantagein its environment. Since A. actinomycetemcomitans is geneticallyclosely related to A. pleuropneumoniae (Dewhirst et al. J. Bacteriol.1992 174:2002-2013) and produces a biofilm similar to that produced byA. actinomycetemcomitans, which as demonstrated herein detaches uponcontact with A. actinomycetemcomitans dispersin B, it is expected thatthe A. pleuropneumoniae DspB homologue identified in SEQ ID NO:10 is afunctionally equivalent or active variant of SEQ ID NO:2, and isincluded in the scope of the present invention. Similarly, sinceActinobacillus ligniersii is genetically closely related toActinobacillus pleuropneumoniae (Dewhirst et al. J. Bacteriol. 1992174:2002-2013) and Haemophilus aphrophilus is genetically closelyrelated to A. actinomycetemcomitans (Dewhirst et al. J. Bacteriol. 1992174:2002-2013; Kaplan et al. J. Clin. Microbiol. 2002 40:1181-1187), andsince both A. ligniersii and Haemophilus aphrophilus produce biofilmssimilar to that produced by A. actinomycetemcomitans, it is expectedthat the Actinobacillus ligiersii and Haemophilus aphrophilus dispersinhomologues identified in SEQ ID NO:4 and SEQ ID NO:8, respectively, arefunctionally equivalent or active variants of SEQ ID NO:2, and areincluded in the scope of the present invention.

The above mentioned examples demonstrate functionally equivalent oractive variants of A. actinomycetemcomitans dispersin B that occur innature. As will be understood by those of skill in the art upon readingthis disclosure, however, artificially produced genes that encodefunctionally equivalent or active variants of A. actinomycetemcomitansdispersin B can also be produced routinely in accordance with theteachings herein using various well known genetic engineeringtechniques. For example, a genetically engineered dispersin B enzymethat lacks 20 N-terminal amino acid residues, and also contained a 32amino acid residue C-terminal tail, which if functionally equivalent tothe natural dispersin B enzyme has been produced. It has also been shownthat the methionine residue at the N-terminus of this geneticallyengineered dispersin B enzyme, when expressed in E. coli, was removed bythe action of methionine aminopeptidase, yet the absence of themethionine did not affect enzyme activity. It has also been shown thatcleavage of the C-terminal 28 amino acid residues from this geneticallyengineered dispersin B enzyme has no affect on enzyme activity. Theseexamples demonstrate that artificial genes can be produced that encodefunctionally equivalent variants of A. actinomycetemcomitans dispersinB. These artificially produced functionally equivalent variants of A.actinomycetemcomitans dispersin B are included in the scope of thepresent invention.

The above mentioned examples demonstrate genetically-engineered,functionally equivalent variants of A. actinomycetemcomitans dispersin Bthat contain either a deletion of amino acid residues at the N-terminusof the protein, or the fusion of an additional polypeptide at theC-terminus of the protein. It is expected that othergenetically-engineered alterations, such as the fusion of an additionalpolypeptide at the N-terminus of the protein, a deletion of amino acidresidues at the C-terminus of the protein, internal deletions andinsertions of amino acid residues, and amino acid substitutions, wouldalso result in functionally equivalent variants of A.actinomycetemcomitans dispersin B. Information about which deletions,insertions, and amino acid substitutions would produce functionallyequivalent variants of A. actinomycetemcomitans dispersin B can beobtained from amino acid sequence alignments, and from commonlyavailable computer software that predicts polypeptide secondarystructures based on both primary amino acid sequences and on amino acidsequence alignments with homologous proteins having knownthree-dimensional structures. A. actinomycetemcomitans dispersin B, forexample, is a member of the family 20 glycosyl hydrolases, a family thatincludes several well-studied enzymes, and a family represented bynumerous homologous primary amino acid sequences in the publicdatabases. In some cases, three-dimensional structures of family 20glycosyl hydrolases are known (Tews et al. Nature Struct. Biol. 19963:638-648). All family 20 glycosyl hydrolases exhibit a (βα)₈-barrelmotif (also known as a TIM-barrel motif; Tews et al. Nature Struct.Biol. 1996 3:638-648; Prag et al. J. Mol. Biol. 2000 300:611-617), whichis by far the most common enzyme fold in the Protein Data Bank (PDB)database of known protein structures. It is estimated that 10% of allknown enzymes have this domain (Wierenge, R. K., FEBS Lett. 2001492:193-198). The (βα)₈-barrel motif is seen in many different enzymefamilies, catalyzing completely unrelated reactions. The availability ofnumerous homologous primary amino acid sequences, combined with theavailability of the three-dimensional structures of several A.actinommycetemcomitans dispersin B homologues, forms the basis of thesesequence alignments and secondary structure predictions. For example,the (βα)₈-barrel motif consists of eight α-helices and eight β-strandssuch that eight parallel β-strands form a barrel on the inside of theprotein, which are covered by eight α-helices on the outside of theprotein. Based on the above mentioned protein sequence alignments andstructural predictions, it is expected that the eight β-strands in A.actinomycetemcomitans DspB comprise the amino acid residues surroundingpositions 41-44, 69-81, 130-134, 169-171, 189-200, 253-256, 288-300, and348-350 of SEQ ID NO:2. Any alteration in the amino acid sequence thatdisrupts the β-strand architecture of these eight regions would beexpected to result in a decrease in enzyme activity because of aconcomitant disruption in the three-dimensional structure of the(βα)₈-barrel of the enzyme. Similarly, based on the above mentionedprotein sequence alignments and structural predictions, it is expectedthat the eight α-helices in A. actinomycetemcomitans DspB comprise theamino acid residues surrounding positions 52-63, 89-93, 143-149,176-183, 214-228, 269-284, 309-321, and 361-374 of SEQ ID NO:2. Anyalteration in the amino acid sequence that disrupts the α-helicalarchitecture of these eight regions would be expected to result in adecrease in enzyme activity because of a concomitant disruption in thethree-dimensional structure of (βα)₈-barrel of the enzyme. Similarly,because the β-strands consist of four inward pointing side chains(pointing into the β-barrel) and four outward pointing side chains(pointing towards the α-helices), it is expected that alterations in theinward-pointing amino acid residues will reduce enzyme activity becauseof concomitant alterations to the substrate binding pocket inside the(βα)₈-barrel, and that alterations in the outward-pointing amino acidresidues will reduce enzyme activity when they interfere with theinteractions between the β-strands and the α-helices. Similarly, theactive site of family 20 glycosyl hydrolases is always located at theC-terminal end of the eight parallel β-strands of the barrel. It isexpected that alterations in the homologous region of A.actinomycetemcomitans dispersin B will affect enzyme activity.Similarly, it is predicted that the introduction of insertions anddeletions into the regions between the α-helices and the β-strands,namely in positions 45-51, 64-68, 82-88, 94-129, 135-142, 150-168,172-175, 182-188, 201-213, 229-252, 257-268, 285-287, 301-308, 322-347,and 351-360, in SEQ ID NO:2, will not effect enzyme activity. Similarly,it is expected that almost any alteration of residues 47 (Arginine), 203(Aspartate) and 204 (Glutamate) will result in complete loss of enzymeactivity, because these three residues have been shown to participatedirectly in substrate binding and catalysis in all family 20 glycosylhydrolases (Mark et al. J. Biol. Chem. 1998 273: 19618-19624; Prag etal. J. Mol. Biol. 2000 300:611-617; Mark et al. J. Biol. Chem. 2001276“10330-10337). Similarly, it is expected that alteration of the threetryptophan residues at positions 236, 257 and 350, to any non-aromaticamino acid residue will result in a decrease in enzyme activity becausethese three tryptophan residues have been shown to line part of thesubstrate-binding pocket that is complementary to the hydrophobicsurfaces of the substrate hexosamine sugar ring (Tews et al. NatureStruct. Biol. 1996 3:638-648). As a result of the locations of theseessential amino acid residues in A. actinomycetemcomitans dispersin B,it is predicted that no more than 46 amino acid residues can be deletedfrom the N-terminus, and no more that 31 amino acids can be deleted fromthe C-terminus, without loss of enzyme activity. All of these geneticalterations that result in functionally equivalent variants are includedin the scope of the present invention.

Genes encoding functionally different variants of A.actinomycetemcomitans dispersin B can also be produced in accordancewith the teachings of the instant application using well known geneticengineering techniques. For example, as mentioned above, it is expectedthat almost any alteration of residues 47 (Arginine), 203 (Aspartate)and 204 (Glutamate) in SEQ ID NO:2 will result in complete loss ofenzyme activity. Alternatively, variants of A. actinomycetemcomitansdispersin B that exhibit characteristics that may be useful in aclinical setting could also be artificially produced. For example, thetemperature optimum of A. actinomycetemcomitans dispersin B is 30° C. Itmay be desirable to produce a genetically-engineered variant ofdispersin B that exhibits a temperature optimum of 37° C., therebyresulting in an increased effectiveness of the enzyme or decreased costof treatment. Such variants can be artificially produced by firstcreating random mutations in the A. actinomycetemcomitans dspB genesequence, for example by using UV light or a chemical mutagen likenitrosoguanidine, and then screening large numbers of these randomvariants, for example in a quantitative 96-well microtiter plate assay(Kaplan et al. J. Bacteriol. 2003 185:4693-4698), for ones that exhibithigher temperature optima. An alternative method is to utilize directedevolution of sequences by DNA shuffling (Christians et al. NatureBiotechnol. 1999 17:259-264; Dichek et al. J. Lipid Res. 199334:1393-1340), combined with a high-throughput robotic screen based upona quantitative 96-well microtiter plate assay (Kaplan et al. J.Bacteriol. 2003 185:4693-4698) to identify variants with increasedtemperature optima. The aforementioned methods can also be used toproduce variants of A. actinomycetemcomitans dispersin B that exhibitincreased substantivity to biomaterials, increased pH optima, increasedstability in aqueous solutions, increased reaction rate, increasedstability upon desiccation, and other characteristics that could resultin increased effectiveness of the enzyme or decreased cost of treatment.An alternative method that can be used to produce useful variants issite-directed mutagenesis. For example, it is expected that the eightα-helices of the (βα)₈-barrel in A. actinomycetemcomi tans dispersin Bcontain many amino acid residues that are exposed on the outer surfaceof the enzyme, and that altering the outward-pointing amino acidresidues of the eight α-helices will alter the outer surface propertiesof the enzyme, thereby potentially increasing the substantivity of theenzyme for biomaterials without affecting enzyme activity. Accordingly,these outward pointing amino acid residues can be systematicallymutated, for example from polar residues to charged residues, and theresulting mutants screened to identify variants with increasedsubstantivity to biomaterials. Functionally different variants of A.actinomycetemcomitans dispersin B that are intended to improve theclinical efficiency or cost effectiveness of the enzyme, when applied todetaching bacterial or fungal cells from biofilms, are included in thescope of the present invention.

Also provided in the present invention are fusion proteins and nucleicacid sequences encoding fusion proteins. Fusion proteins of the presentinvention comprise an amino acid sequence for an isolated soluble,β-N-acetylglucosaminidase protein which promotes detachment of bacterialcells from a biofilm and a second polypeptide. Exemplary secondpolypeptides of these fusion proteins include, but are not limited to,those which facilitate purification such as a His tag, those whichfacilitate attachment to a surface such as an antibody or a protein suchas albumin, fibronectin or thrombin, and/or those which target theenzyme to the surface of bacterial or fungal cell such as a specificbacterial or fungal receptor. Nucleic acid sequences encoding suchfusion proteins comprise an isolated nucleic acid sequence encodingsoluble, β-N-acetylglucosaminidase or an active fragment or variantthereof which promotes detachment of bacterial or fungal cells from abiofilm and a second nucleic acid sequence encoding a secondpolypeptide. In a preferred embodiment, the second nucleic acid sequenceencodes a polypeptide such as a His tag which facilitates purification,an antibody or protein such as albumin, fibronectin or thrombin whichfacilitates attachment of the fusion protein to a surface, or abacterial or fungal receptor which specifically targets the fusionprotein to the surface of a bacterial or fungal cell, respectively.

The dispersin B protein engineered to contain an octahistidine metalbinding site at its C-terminus was expressed in E. coli. The protein waspurified by Ni-affinity chromatography and the dispersin B portion wascleaved from the hybrid protein using thrombin. Analysis of the purifiedcleaved dispersin B protein by SDS-PAGE revealed the protein to migratewith an apparent molecular mass of 41 kDa. The N-terminal sequence ofdispersin B was XCVKGNSIYPQK (SEQ ID NO:11) (where X is an unidentifiedresidue). This matched codons 22 to 33 of CU1000 dspB, thus indicatingthat the dipeptide Met-Asn was cleaved from the N-terminus of thedispersin B fusion protein when expressed in E. coli. Analysis ofpurified, cleaved dispersin B protein by mass spectrophotometry resultedin a single major peak with an apparent molecular mass of 41.5 kDa,consistent with the predicted molecular mass of 41.4 kDa for the cleavedand processed dispersin B protein. The yield of dispersin B expressed inB. coli was 30 mg of protein per liter of culture.

The ability of dispersin B to cleave the glycosidic linkages of various4-nitrophenyl-labeled synthetic hexosamine substrates was tested in anin vitro enzyme assay. Dispersin B showed specificity for the 1→4glycosidic bond of β-substituted N-acetylglucosaminide, consistent withthe known functions of other family 20 glycosyl hydrolases (Tews et al.Nature Struct. Biol. 1996 3:638-648). Dispersin B showed no activityagainst α-substituted N-acetylglucosaminide, or against α- orβ-substituted N-acetylgalactosamine.

The glycosyl hydrolase activity of dispersin B was optimal at pH 5.0,which is similar to the pH optima of other family 20 glycosylhydrolases. Dispersin B displayed maximum activity at 30° C. Dispersin Bglycosyl hydrolase activity was inhibited by quinacrine (Kovacs, P. andCsaba, G. Cell Biochem. Funct. 2001 19:287-290) and NAG-thiazoline (Market al. J. Biol. Chem. 2001 276:10330-10337), two small moleculeinhibitors of family 20β-N-acetylglucosaminidases.

The effects of dispersin B protein on biofilm cell detachment of A.actinomycetemcomitans mutant strain JK1023 were then examined. In theseexperiments, dispersin B protein was added to growth medium of mutantstrain JK1023 to determine if addition of this protein restored releaseof cells into the medium and dispersion. Polystyrene rods containingbiofilm colonies of strain JK1023 were suspended in broth containingvarious amount of dispersin B, and the amount of biofilm cell detachmentwas measured by staining the bacteria growing on the bottom of the wellwith crystal violet. Purified dispersin B restored the ability of mutantstrain JK1023 to release cells into the medium and colonize the bottomof the microtiter plate well in a dose-dependent manner.Heat-inactivated dispersin B had no effect on biofilm cell detachment ofstrain JK1023.

The effects of dispersin B protein on detachment of preformed biofilmcolonies of A. actinomycetemcomitans and other bacteria were alsoexamined. In these experiments, addition of dispersin B caused thedetachment of preformed biofilm colonies of wild-type strain CU1000.Dispersin B (50 μg/ml) caused a 90% reduction in the amount ofsurface-associated bacteria after 6 hours. Further, analysis by lightmicrography showed that the surface of treated colonies became grainyand flocculent when compared to the smooth-textured biofilm coloniesobserved with mock-treated cells. Also, the surface of the culturevessel became covered with a similar grainy material which had a fibrousappearance under higher power. These findings are consistent with theobserved reduction in adherence of preformed biofilm colonies treatedwith dispersin B.

Dispersin B caused a similar reduction in biofilm density when testedagainst biofilm colonies of four phylogenetically diverse strains of A.actinomycetemcomitans representing four different serotypes, a strain ofthe closely related bacterium Haemophilus aphrophilus, and two strainsof the swine pathogen Actinobacillus pleuropneumoniae. Dispersin B didnot cause the detachment of biofilm colonies of Neisseria subflava,Cardiobacterium hominis or Streptococcus mitis, bacteria which do nothave biofilms comprising N-acetyl glucosamine residues.

Dispersin B also causes the detachment of Staphylococcus epidermidisfrom surfaces. The Gram-positive bacterium S. epidermidis is the mostcommon cause of infection associated with catheters and other indwellingmedical devices. S. epidermidis produces an extracellular slime composedof a polysaccharide containing primarily N-acetylglucosamine residues(Mack et al. J. Bacteriol. 1996 178:175; Baldassarri et al. Infect.Immun. 1996 64:3410) which enables it to form adherent films on plasticsurfaces. Biofilm bacteria such as S. epidermidis are highly resistantto antibiotics and host defenses and nearly impossible to irradicate(Costerton et al. Annu. Rev. Microbiol. 1995 49:711). Thus, attachmentof this bacteria to indwelling devices such as catheters can lead toosteomyelitis, acute sepsis and death, particularly in immunocompromisedpatients, and is a leading cause of nosocomial bloodstream andcardiovascular infections as well as morbidity in hospitalized patients(Vuong, C. and Otto, M. Microbes, Infect. 2002 4:481).

Four different strains of S. epidermidis isolated from infectedintravenous catheters were used in these experiments. All four strainscontained the ica genetic locus and produced dark red colonies on Congored agar, both of which are indicative of slime production (Aricola etal. J. Clin. Microbiol. 2001 39:2151; Aricola et al. Biomaterials 2002Biomaterials 23:4233). The ability of the four strains to form biofilmswas measured by making serial dilutions of overnight cultures in freshbroth and then transferring the dilutions to the wells of a 96-wellpolystyrene microliter plate. After 16 hours of incubation, the wellswere washed under running tap water to remove loosely adherent cells andthe bacteria remaining attached to the bottoms of the well were stainedwith crystal violet. As expected all four strains produced adherentbiofilms as indicated by the presence of dark-staining material on thebottoms of the wells. The amount of dark-staining material wasquantitated by measuring its optical density at 590 nm in a microliterplate reader. When dispersin B protein was added to the wells 30 minutesprior to washing (final concentration, 40 μg/ml) little or no biofilmmaterial was evident. In contrast, heat inactivated dispersin B proteinhad no effect on S. epidermidis biofilms. Two otherN-acetylglucosaminidase enzymes that are homologous to A.actinomycetemcomitans dispersin B, Serratia marcescens chitinase andjack bean β-hexosaminidase, also had no effect on S. epidermidisbiofilms. Unlike the orthologs described herein, these homologousproteins share less than 25% identity with dispersin B and do notexhibit biofilm-releasing activity. Thus, these experiments aredemonstrative of dispersin B enzymatic activity being responsible forremoving S. epidermidis biofilm cells from the surfaces of the wells.Dispersin B had no effect on viability of S. epidermidis cells.

The amount of dispersin B protein and the length of time needed toremove S. epidermidis biofilms from the microliter plate wells were alsoexamined. In these experiments, multiple wells were inoculated with a10⁻⁴ dilution of a S. epidermidis culture and the plate was incubatedfor 16 hours. After washing away loosely adherent cells, the wells werefilled with phosphate buffered saline (PBS) and then various amounts ofdispersin B protein (200 Pg to 120 μg per ml final concentrations) wereadded to the wells for various lengths of time (0 to 9 minutes).Dispersin B treatment at a concentration of 4.8 μg/ml resulted in adecrease in absorbance to background levels (ca. 0.09 O.D. units) after2 minutes. At a concentration of 40 ng/ml, dispersin B resulted in agreater than 50 percent reduction in optical density after 9 minutes(from 3.63 to 1.74 O.D. units. These data demonstrate that dispersin Bcauses detachment of S. epidermidis biofilms of clinically achievableconcentrations of the enzyme.

Biofilm cell detachment was quantitated by growing S. epidermidisbiofilms on polystyrene rods and then transferring the rods to tubescontaining PBS (as a control) or PBS with 60 μg/ml of dispersin B. Thetubes were incubated for 15 minutes, rinsed in PBS, and the bacteriaremaining attached to the rods after treatment were removed bysonication and then quantitated by plating serial dilutions of thesonicates on agar. Mock-treated and dispersin B-treated rods werecompared after staining with crystal violet. The mock-treated controlrod contained a layer of dark-staining material corresponding to thethick biofilm that formed on its surface. The dispersin B-treated rodshowed no trace of dark-staining material and was similar in appearanceto a rod which was sonicated prior to staining and to an uninoculatedrod. Quantitation of cells remaining attached to the rods revealed thatdispersin B treatment resulted in a 5.8 log reduction in the number ofsurface-associated bacteria.

The ability of dispersin B to remove S. epidermidis biofilms grownattached to polyurethane and Teflon intravenous catheters was alsoexamined. In these experiments, catheters were placed in tubescontaining a 10⁻³ dilution of a S. epidermidis culture and incubated for16 hours. The catheters were then rinsed with PBS and transferred totubes containing PBS (as a control) or PBS with 60 μg/ml of dispersin B.After 5 minutes the catheters were rinsed with PBS and the biofilmbacteria remaining attached to the surface were stained with methyleneblue (for polyurethane catheters) or crystal violet (for Tefloncatheters). The control catheters contained a layer of dark-stainingmaterial on their surfaces indicating the presence of a biofilm, whereasthe dispersin B-treated catheters contained no dark-staining materialand were similar in appearance to uninoculated catheters.

Thus, dispersin B of the present invention is capable of removing S.epidermidis biofilms from various plastic biomaterials.

The ability of precoating surfaces with dispersin B to prevent S.epidermidis biofilm formation was also demonstrated. In theseexperiments, polyurethane and Teflon catheters in tubes containing PBSor PBS with 40 μg/ml of dispersin B were incubated at 4° C. for 24hours. The catheters were then rinsed with PBS and transferred to tubescontaining a 10⁻¹ dilution of a S. epidermidis culture After 6 hours,the catheters were rinsed with PBS to remove loosely adherent cells andthen stained as described supra. The surfaces of control catheters werecovered with a layer of dark-staining material indicating the presenceof a biofilm, whereas the surfaces of dispersin B-coated catheterscontained no dark-staining material and were similar in appearance touninoculated catheters. As shown, precoating plastic catheters withdispersin B of the present invention significantly reduced S.epidermidis attachment or biofilm formation. Catheters that wereprecoated for 10 minutes, and catheters that were precoated for 24 hoursand then dried, were also resistant to colonization and biofilmformation by S. epidermidis.

Thus, as demonstrated by these experiments, addition of an isolateddispersin B protein as well as mutation of the dspB gene modulates thedetachment of cells from biofilm colonies of various bacteria,particularly bacteria with a biofilm comprising a polysaccharidecontaining N-acetylglucosamine. Fungi also form biofilms of clinicalsignificance which may compromise polysaccharide containingN-acetylglucosamine. It is believed that dispersin B will also beeffective in degrading these fungal polysaccharides and modulatingdetachment of such fungal cells from their biofilms.

Accordingly, the present invention also relates to methods formodulating detachment of bacterial or fungal cells from biofilms,particularly bacteria or fungal with a biofilm comprising apolysaccharide containing N-acetylglucosamine.

By “modulating detachment” as used herein it is meant to be inclusive ofincreases as well as decreases in bacterial or fungal biofilm detachmentor release of bacterial or fungal cells from the biofilm. Further, by“modulating detachment” it is also meant to be inclusive of changes inthe ability of the bacteria or fungal to attach as a biofilm. Forexample, as demonstrated herein, dispersin B modulates detachment of S.epidermidis not only by promoting detachment but also by inhibiting theability of the bacteria to attach to surfaces and form a biofilm.

In one embodiment of the present invention, the method comprisesmutating dspB of bacterial cells to inhibit detachment of bacterialcells from biofilms such as in the JK1023 mutant of the presentinvention. In another embodiment, the method comprises decreasingexpression and/or levels of soluble, β-N-acetylglucosaminidase orinhibiting activity of soluble, β-N-acetylglucosaminidase in bacterialcells so that detachment of bacterial cells is decreased.

The present invention also provides methods for promoting detachment ofbacterial or fungal cells from a biofilm which comprises contactingbacterial or fungal cells with soluble, β-N-acetylglucosaminidase or anactive fragment or variant thereof or a nucleic acid sequence encodingsoluble, β-N-acetylglucosaminidase or an active fragment or variantthereof. For example, A. actinomycetemcomitans dispersin B was found todetach biofilms of Haemophilus aphrophilus, Actinobacilluspleuropneumonaie and S. epidermidis. It is believed that biofilmdetachment of Actinobacillus ligniersii, as well as other bacteria orfungi with a biofilm comprising a polysaccharide containingN-acetylglucosamine including, but in no way limited to, Staphylococcusaureus and Yersinia pestis will also be promoted in the presence ofsoluble β-N-acetylglucosaminidase or an active fragment thereof of thepresent invention.

Accordingly, isolated dispersin B proteins and active fragments orvariants thereof can be used to prevent or inhibit bacterial or fungalbiofilm attachment and to treat infections by such bacteria or fungi.

In one embodiment, the isolated dispersin B protein or active fragmentor variant thereof is used directly as a parenteral to treat biofilminfections such as mastitis in ewes, intramammary infections in cows orosteomyelitis and infective endocarditis in humans. In this embodiment,the isolated soluble, β-N-acetylglucosaminidase protein or activefragment or variant thereof is preferably administered as apharmaceutical composition in a pharmaceutically acceptable carrier toan organism.

By “organism”, as used herein it is meant to be inclusive of all animalsincluding, but not limited to mammals, and most preferably humans.

Any pharmaceutically acceptable vehicle or carrier, as well as adjuvant,can be used in the manufacture, dissolution and administration ofpharmaceutical preparations comprising dispersin B protein or activefragment or variant thereof. Such vehicles, carriers and adjuvants arewell known to those of skill in the art and described in text books suchas Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985. Appropriate concentrations of active composition to beincorporated into pharmaceutical compositions can be routinelydetermined by those skilled in the art and is dependent upon the form ofadministration as well as the severity of the condition being treated.

Pharmaceutical formulations suitable for oral administration may beprovided in convenient unit forms including, but not limited to,capsules or tablets, each containing a predetermined amount of thedispersin B protein or active fragment or variant thereof; as a powderor granules; as a solution, a suspension or as an emulsion. Thedispersin B protein or active fragment or variant thereof can also bepresented as a bolus, electuary, or paste. Tablets and capsules for oraladministration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Thetablets may be coated according to methods well known in the art. Timedrelease formulations, which are known in the art, may also be suitable.Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicles before use. Such liquid preparations may contain conventionaladditives such as suspending agents, non-aqueous vehicles, includingedible oils, or preservatives.

Dispersin B protein or active fragments or variants thereof of thepresent invention may also be formulated for parenteral administration,such as by injection, for example bolus injection or continuousinfusion, and may be provided in unit dose form in ampules, pre-filledsyringes, small volume infusion or in multi-dose containers with anadded preservative. Pharmaceutically acceptable compositions comprisinga dispersin B protein or active fragment or variant thereof forparenteral administration may be in the form of a suspension, solutionor emulsion in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.Alternatively, the active ingredient may be in powder form, obtained byasceptic isolation of sterile solid or by lyophilization from solution,for constitution with a suitable vehicle such as sterile, pyrogen freewater, before use.

For topical administration to the epidermis, dispersin B protein or anactive fragment or variant thereof of the present invention may beformulated in an ointment, cream, or lotion, or as a transdermal patch.Ointments and creams, may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, suspending agents, thickening agents, or coloring agents.Formulations suitable for topical administration in the mouth includelozenges comprising dispersin B protein or an active fragment or variantthereof in a flavored base, usually sucrose and acacia or tragacanth;pastilles comprising the active ingredient in an inert base such asgelatin and glycerin or sucrose and acacia; and mouth washes comprisingthe active ingredient in a suitable liquid carrier. For topicaladministration to the eye, the dispersin B protein or active fragment orvariant thereof can be made up in solution or suspension in a suitablesterile aqueous or non-aqueous vehicle. Additives such as buffers (e.g.sodium metabisulphite or disodium edeate) and thickening agents such ashypromellose can also be included.

For intra-nasal administration, dispersin B protein or an activefragment or variant thereof of the present invention can be provide in aliquid spray or dispersible powder or in the form of drops. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilizing agents, or suspending agents.Liquid sprays are conveniently delivered from pressurized packs.

For administration by inhalation, dispersin B protein or active fragmentor variant thereof of the present invention can be delivered byinsufflator, nebulizer or a pressurized pack or other convenient meansof delivering the aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thedispersin B protein or active fragment or variant thereof of the presentinvention can take the form of a dry powder composition, for example apowder mix of the active component and a suitable powder base such aslactose or starch. The powder composition may be presented in unitdosage form in, for example, capsules, cartridges or blister packs ofgelatins, from which the powder can be administered with the aid of aninhalator or insufflator.

When desired, any of the above-described formulations may be adapted toprovide sustained release of the dispersin B protein or active fragmentor variant thereof.

The amount of dispersin B protein or active fragment or variant thereofof the present invention required for use in treatment will of coursevary not only with the particular protein or active fragment or variantselected but also with the route of administration, the nature of thecondition being treated, and the age and condition of the organism.

Increasing detachment of bacteria from a biofilm is also expected todecrease resistance of the bacteria to antibiotic therapy. Accordingly,the present invention also provide methods for enhancing efficacy ofantibiotic therapy against bacterial infections by administration of apharmaceutical composition of the present invention in combination withor prior to administration of an antibiotic.

In another embodiment of the present invention, wound dressingsincluding but not limited to sponges or gauzes can be impregnated withthe isolated dispersin B protein or active fragment or variant thereofto prevent or inhibit bacterial or fungal attachment and reduce the riskof wound infections. Similarly, catheter shields as well as othermaterials used to cover a catheter insertion sites can be coated orimpregnated with a dispersin B protein or active fragment or variantthereof to inhibit bacterial or fungal biofilm attachment thereto.Adhesive drapes used to prevent wound infection during high risksurgeries can be impregnated with the isolated protein or activefragment or variant thereof as well. Additional medical devices whichcan be coated with a dispersin B protein or active fragment or variantthereof include, but are not limited, central venous catheters,intravascular catheters, urinary catheters, Hickman catheters,peritoneal dialysis catheters, endotracheal catheters, mechanical heartvalves, cardiac pacemakers, arteriovenous shunts, schleral buckles,prosthetic joints, tympanostomy tubes, tracheostomy tubes, voiceprosthetics, penile prosthetics, artificial urinary sphincters,synthetic pubovaginal slings, surgical sutures, bone anchors, bonescrews, intraocular lenses, contact lenses, intrauterine devices,aortofemoral grafts and vascular grafts. Exemplary solutions forimpregnating gauzes or sponges, catheter shields and adhesive drapes orcoating catheter shields and other medical devices include, but are notlimited to, phosphate buffered saline (pH approximately 7.5) andbicarbonate buffer (pH approximately 9.0).

In yet another embodiment, an isolated dispersin B protein or activefragment or variant thereof can be incorporated in a liquid disinfectingsolution. Such solutions may further comprise antimicrobials orantifungals such as alcohol, providone-iodine solution and antibioticsas well as preservatives. These solutions can be used, for example, asdisinfectants of the skin or surrounding area prior to insertion orimplantation of a device such as a catheter, as catheter lock and/orflush solutions, and as antiseptic rinses for any medical deviceincluding, but not limited to catheter components such as needles,Leur-Lok connectors, needleless connectors and hubs as well as otherimplantable devices. These solutions can also be used to coat ordisinfect surgical instruments including, but not limited to, clamps,forceps, scissors, skin hooks, tubing, needles, retractors, scalers,drills, chisels, rasps and saws.

The nucleic acid and amino acid sequences of the present invention, aswell as the mutant JK1023 strain can also be used to identify agentswhich modulate detachment of bacterial or fungal cells from biofilms.For example, the ability of an agent to modulate activity and/orexpression of soluble, β-N-acetylglucosaminidase of the presentinvention can be assessed.

Examples of such agents include, but are not limited to antisenseoligonucleotides or ribozymes targeted to the dspB gene, peptidomimeticsof dispersin B, and small organic chemicals such as quinacrine andNAG-thiazoline which modulate dispersin B activity and/or levels and/orexpression.

Agents which inhibit the ability of soluble, β-N-acetylglucosaminidaseto promote detachment of bacterial cells from biofilms are expected tobe useful in preventing the dissemination of infectious bacteria,particularly infectious bacteria of the oral cavity such as A.actinomycetemcomitans and closely related bacterium such as Haemophilusaphrophilus.

Agents which mimic dispersin B activity such as peptidomimetics andsmall organic molecules similar in structure and activity to dispersin Bcan be used in similar fashion to isolated dispersin B or an activefragment or variant thereof to prevent, inhibit or treat infectionresulting from bacterial or fungal biofilm attachment to surfaces. Suchuses are described herein in detail supra.

The present invention also provides primer pairs and kits comprisingsuch primer pairs for use in identifying additional species of bacteriawith dispersin B homologues. An exemplary degenerate primer pair usefulin the kits of the present invention comprises 5′-GAYCAYGARAAYTAYCG-3′(SEQ ID NO:12) and 5′-TCNCCRTCRTARCTCCA-3′ (SEQ ID NO:13), where Y is Cor T, and R is A or G. Kits of the present invention preferably furthercomprise instructions for use of the kit and/or positive and negativecontrol samples. Bacteria identified by these kits as having a dispersinB homologue can be further examined to determine if the homolog is anortholog exhibiting the same or similar enzymatic activity as dispersinB. The primers and kits of the present invention are thus useful inidentifying additional bacteria, biofilm attachment of which can bemodulated using the nucleic acid sequences, amino acid sequences, andagents described herein as well as additional orthologous nucleic acidsequences and amino acid of dispersin B.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Bacterial Strains and Growth Conditions

A. actinomycetemcomitans CU1000 (serotype f) is a clinical strainisolated from a 13 year old patient with localized juvenileperiodontitis (Fine et al. Microbiol. 1999 145:1335-1347). StrainCU1000N is a spontaneous nalidixic acid derivative of strain CU1000 thatdisplays the same surface attachment, biofilm colony formation andbiofilm dispersal phenotypes as the parental strain (Fine et al. Arch.Oral Biol. 2001 46:1065-1078; Kachlany et al. J. Bacteriol. 2000182:6169-6176; Kachlany et al. Mol. Microbiol. 2001 40:542-554; Thomsonet al. J. Bacteriol. 1999 181:7298-7307). Mutagenesis of strain CU1000Nwith transposon IS903φkan was carried in accordance with the proceduresset forth by Thomson et al. (J. Bacteriol. 1999 181:7298-7307). Otherstrains utilized include A. actinomycetemcomitans DF2200 (serotype a),NJ8800 (serotype b), NJ2700 (serotype c), and NJ9500 (serotype e)(Kaplan et al. J. Clin. Microbiol. 2002 40:1181-1187); and A.actinomycetemcomitans strain IDH781 (Saarela et al. Oral Microbiol.Immunol. 1993 8:111-115); Haemophilus aphrophilus NJ8700 (Kaplan et al.J. Clin. Microbiol. 2002 40:1181-1187); Neisseria subflava NJ9702(Kaplan, J. B. and Fine, D. H. Appl. Environ. Microbiol. 200268:4943-4950); Cardiobacterium hominis NJ6500; Actinobacillus ligniersiistrain 19393 (obtained from ATCC, Manassa, Va.); and Streptococcus mitisNJ9705 (Kaplan, J. B. and Fine, D. H. Appl. Environ. Microbiol. 200268:4943-4950). S. epidermidis strains were isolated from the surfaces ofinfected intravenous catheters and were identified by using theApi-Staph biochemical identification kit (Biomérieux, Lyons France). A.pleuropneumoniae strains were obtained from the Veterinary DiagnosticsLaboratory (Iowa State University, Ames, Ill.). Bacteria were grown inTrypticase soy broth (BD Biosystems) supplemented with 6 grams of yeastextract and 8 grams of glucose/liter. Inoculated culture vessels wereincubated at 37° C. in 10% CO₂, except for S. epidermidis cultures,which were incubated at 37° C. in air.

Example 2 Cloning and Sequencing dspB

The transposon insertion site in A. actinomycetemcomitans mutant strainJK1023 was cloned and sequenced by using an inverse PCR method inaccordance with Kaplan et al. (Infect. Immun. 2001 69:5375-5384). TheDNA sequence of the inverse PCR product was compared to the genomesequence of A. actinomycetemcomitans strain HK1651 from theActinobacillus Genome Sequencing Project and the transposon was found tohave inserted into a long open reading frame (ORF) which was designateddspb. Primers that hybridize to sequences upstream and downstream fromHK1651 dspB were used to amplify by PCR the dspB coding region from A.actinomycetemcomitans strain CU1000 using methods in accordance withKaplan et al. (Infect. Immun. 2001 69:5375-5384). The forward primer(5-GCGCGCCAatgAATTGTTGCGTAAAAGGCAATTCC-3 (SEQ ID NO:14)) introduced anNdeI restriction site (underlined) and an ATG initiation codon (lowercase) at codon positions 19 to 20 of dspB, and the reverse primer(5-GCGGTACCCTCATCCCCATTCGTCTTATGAATC-3 (SEQ ID NO:15)) replaced the dspBstop codon with a KpnI restriction site (underlined). The PCR product(1,106 bp) was digested with NdeI and KpnI and ligated into theNdeI/KpnI sites of plasmid pET29b (Novagen). The insert of the resultingplasmid (designated pRC1) was subjected to DNA sequence analysis inaccordance with procedures described by Kaplan et al. (Infect. Immun.2001 69:5375-5384).

Example 3 Expression and Purification of Recombinant

Dispersin B Protein

Plasmid pRC1 carries a gene that encoded amino acids 21 to 381 of dspBfused to a 32 amino acid residue C-terminal tail containing anhexahistidine metal-binding site and a thrombin protease cleavage sitewhich could be used to cleave the C-terminal tail from the hybridprotein. This gene was located downstream from anisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promoter.Expression of DspB in E. coli

A one liter Erlenmeyer flask containing 500 ml of LB broth supplementedwith 50 μg/ml of kanamycin was inoculated with 5 ml of an overnightculture of E. coli strain BL21(DE3) (Dubendorff, J. W. and Studier, F.W. J. Mol. Biol. 1991 219:61-68) transformed with pRC1. The flask wasincubated at 37° C. with agitation (200 rpm) until the optical densityof the culture (measured at 280 nm) reached 0.6 (approximately 3 hours).IPTG was added to a final concentration of 0.2 mM and the flask wasincubated for an additional 5 hours with agitation. The cells wereharvested by centrifugation for 15 minutes at 6,000×g and the cellpellet was stored at −80° C.

Protein Purification

The cell pellet was thawed on ice and resuspended in 20 ml of lysisbuffer [20 mM Tris-HCl (pH 7.2), 0.1% sodium dodecyl sulfate] containing10 mg/ml lysozyme. The cell suspension was sonicated for 30 seconds at50% capacity, 70% duty cycle in a Branson model 4550 sonicator equippedwith a microprobe and then cooled on ice for 30 seconds. The sonicationand cooling steps were repeated four additional times. The cells werepelleted by centrifugation as above and the supernatant was transferredto a new tube. The cell pellet was resuspended in 20 ml of lysis bufferwithout lysozyme and five additional cycles of sonication and coolingwere performed. The cells were pelleted by centrifugation and thesupernatant was removed and transferred to a new tube. The twosupernatants were combined and loaded onto a 3 ml bed volume Ni-affinitycolumn (catalog no. 154-0990, Pharmacia) according to the instructionssupplied by the manufacturer. The column was washed with 50 ml of washbuffer [50 mM MOPS (pH 8.5), 20 mM KCl] containing 5 mM imidazole,followed by 25 ml of wash buffer containing 50 mM imidazole and 25 ml ofwash buffer containing 100 mM imidazole. Fractions (1.5 ml each) werecollected during the final wash and assayed for the presence of thehybrid protein by SDS polyacrylamide gel electrophoresis (SDS-PAGE) andCoomassie blue staining in accordance with procedures described bySambrook et al. (1989. Molecular cloning: a laboratory manual, 2nd ed.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Fractions containing the protein were pooled and dialyzed overnightagainst water using a 10,000 MW cut-off dialysis membrane. The purifiedprotein was digested with 5 units of thrombin (Novagen) per mg ofprotein for 1 hour at room temperature and the thrombin was removedusing a Thrombin Cleavage Capture Kit (Novagen) according toinstructions supplied with the kit. Undigested protein was removed byloading the sample onto a Ni-affinity column as described above andwashing the column with 10 ml of wash buffer containing 5 mM imidazole.Fractions of the wash (1.5 ml each) were collected and analyzed for thepresence of the protein by SDS-PAGE. Fractions containing the proteinwere pooled, dialyzed against water, and stored at −20° C. N-terminalsequence analysis of the purified protein was carried out using theEdman degradation procedure on a Beckman model 2300 protein sequencer.Mass spectra were determined by using a Hitachi model 4414 massspectrometer.

Example 4 Enzyme Assays

Synthetic substrates (purchased from Sigma Chemical Co.) were4-nitrophenyl-N-acetyl-β-D-galactosaminide,4-nitrophenyl-N-acetyl-α-D-galactosaminide,4-nitrophenyl-N-acetyl-β-D-glucosaminide, and4-nitrophenyl-N-acetyl-α-D-glucosaminide. Enzyme reactions were carriedout in a 10 ml volume containing 50 mM sodium phosphate buffer (pH 5.9),100 mM NaCl, 5 mM substrate, and 3.7 μg/ml purified protein in a 15 mlpolypropylene tube placed in a 37° C. water bath. The reaction wasterminated at various times by transferring 1 ml of the reaction mixtureto a new tube containing 5 μl NaOH. The increase in absorption resultingfrom the release of p-nitrophenolate in each tube was measured in aShimadzu UV-Mini spectrophotometer set to 405 nm.

Example 5 Identification of dspB Orthologues in Other Strains of A.actinomycetemcomitans and in Other Species of Bacteria

The microbial genome database www.ncbi.nlm.nih.gov was searched forhomologues of A. actinomycetemcomitans dspB. dspb homologues wereidentified in the unfinished genomes of A. pleuropneumoniae serovars 1,5 and 7. The A. pleuropneumoniae dspB homologues displayed approximately60% identity at the amino acid level with the A. actinomycetemcomitansCU1000 DspB sequences. Additional searching was performed for DspBhomologues in other members of the Pasteurellaceae family. The aminoacid sequence of A. actinomycetemcomitans CU1000 DspB was aligned withthe A. pleuropneumoniae DspB homologues and two regions of the sequencewere identified that were highly conserved. Degenerate oligonucleotideprimers were then synthesized that hybridized to DNA sequences encodingthese conserved amino acids (5′-GAYCAYGARAAYTAYCG-3′ (SEQ ID NO:12) and5′-TCNCCRTCRTARCTCCA-3′ (SEQ ID NO:13), where Y=C or T, R=A or G, andN=A or C or G or T) and these primers were used to amplify by PCRgenomic DNAs purified from various species of Pasteurellaceae. A PCRproduct of the expected size was observed in genomic DNA from A.actinomycetemcomitans strain IDH781 (Saarela et al. 1993. OralMicrobiol. Immunol. 8:111-115), A. pleuropneumoniae strain IA5 (obtainedfrom the Veterinary Diagnostics Laboratory, Iowa State University, Ames,Iowa), Haemophilus aphrophilus strain NJ8700 (Kaplan et al. 2002 J.Clin. Microbiol. 40:1181-1187), and A. lignieresii strain 19393(obtained from the American Type Culture Collection, Manassas, Va.). NoPCR product was observed with DNA from Haemophilus somnus,Actinobacillus equuli, Pasteurella multocida, and Mannheimiahaemolytica.

The PCR products were cloned into multicopy plasmids and subjected toDNA sequence analysis. FIG. 1 shows a comparison of the predicted DspBamino acid sequence of A. actinomycetemcomitans CU1000 DspB and thesequences of the DspB homologues from the other strain of A.actinomycetemcomitans and other Pasteurellaceae bacteria.

Example 6 Overexpression of dspB in a Wild-type Strain of A.actinomycetemcomitans

In order to determine the effects of overexpressing dspb in a wild-typestrain of A. actinomycetemcomitans, a plasmid was constructed whichcontains dspb under the control of anisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. Thisplasmid was introduced into wild-type strain CU1000, and the cells weregrown in the presence of 1 mM IPTG. CU1000 cells harboring the dspBexpressing plasmid exhibited a smooth-colony morphology on agar andproduced biofilm colonies in broth that displayed a hyper-dispersingphenotype, as indicated by the presence of increased numbers ofsatellite colonies on the surface of the culture vessel. These findingsconfirm that dspB expression parallels the amount of biofilm dispersal.

Example 7 Detachment of Biofilm Cells from Polystyrene Rods inMicrotiter Plates

An assay to measure the detachment of cells from preformed biofilmcolonies grown on polystyrene rods was carried out in 96-well microtiterplates. Biofilm colonies were grown on polystyrene rods suspended inbroth in the 96-wells of a microtiter plate. Cells that detached fromthe biofilm fell to the bottom of the well where they attached to thesurface and formed new biofilm colonies. The amount of biofilm growth onthe bottom of the well, which was proportional to the Number of cellsthat detached from the biofilm colonies on the rods, was measured bystaining with crystal violet. The detachment assay was carried out asfollows.

Construction of the Apparatus

The lid of a 96-well polystyrene flat-bottomed tissue culture plate(Falcon No. 353072) was modified as follows: First, 96 1.5-mm diameterholes were drilled in the lid, with each hole in a positioncorresponding to the center of one of the 96 wells. Then, an 11-mm longpolystyrene rod (1.5-mm diameter, Plastruct Corp., City of Industry, CA)was placed in each hole (with one end of the rod flush against the topof the lid) and secured with trichloromethane plastic solvent. When thismodified lid was placed on a 96-well microtiter plate bottom, the rodswere suspended in the wells with the bottom of each rod approximately 2mm above the bottom of the well. The modified lid was sterilized bysoaking in 70% ethanol for 30 minutes and air drying in a biologicalsafety cabinet.

Inoculation and Incubation of Polystyrene Rods

The microtiter plate bottom was filled with medium (100 μl per well) andeach well was inoculated with a single 2-3 day old colony from an agarplate using a sterile toothpick. The modified lid was then placed on theinoculated plate to submerge the polystyrene rods in the inoculatedmedium, and the plate was incubated at 37° C. for 24 hours to allow thatbacteria to adhere to the rods. The lid was then transferred to a freshmicrotiter plate containing prewarmed medium and incubated for anadditional 24 hours to allow biofilm cells to detach from the rods.

Measuring Detached Cells

The lid was removed and the plate was washed extensively under runningtap water to remove loosely adherent cells. The wells were filled with100 μl of Gram-staining reagent (2 grams crystal violet, 0.8 gramsammonium oxalate, 20 ml ethanol per 100 ml) and the plate was incubatedat room temperature for 10 minutes. The plate was re-washed extensivelyunder running tap water to remove unbound dye. The wells were thanfilled with 100 μl of ethanol and the plate was incubated at roomtemperature for 10 minutes to solubilize the dye. The optical density(at 590 nm) of the ethanol/dye solution in each well was measured usinga Bio-Rad benchmark microplate reader.

Example 8 Growth of Biofilms on Polystyrene Rods

Polystyrene rods (1.5 mm diam; Plastruct Corp., City of Industry,Calif.) were cut into 35 mm lengths, sterilized in 70% ethanol for 30minutes, and air dried in a biological safety cabinet. Rods were placedinto 1.5 ml microcentrifuge tubes containing 0.5 ml of broth inoculatedwith S. epidermidis and incubated for 16 hours. Rods were then rinsedunder running tap water and then placed in fresh microcentrifuge tubescontaining 0.5 ml of PBS or PBS plus dispersin B. Rods were rinsed withwater and stained with crystal violet as previously described (Kaplan,J. B., and Fine, D. H. Appl. Environ. Microbiol. 2002 68:4943-4950). Forsonication, rods were placed in 15 ml conical centrifuge tubescontaining 3 ml of PBS at then sonicated for 30 seconds at 40% dutycycle and 70% capacity in Branson model 200 sonicator equipped with acup horn. For quantitation of detached cells, sonicates were seriallydiluted and plated on medium solidified with 1.5% agar.

Example 9 Growth of Biofilms in Polystyrene Microtiter Plates

The wells of a 96-well polystyrene microtiter plate (model 3595,Corning) were filled with 100 μl of broth inoculated with S. epidermidisand the plate was incubated for 16 hours. Microtiter plates were washedby aspirating the medium and washing the well three times with 200 μl ofPBS, or by submerging the entire plate in a tub of cold, running tapwater. Biofilms were stained with crystal violet as previously described(Kaplan, J. B., and Fine, D. H. Appl. Environ. Microbiol. 200268:4943-4950).

Example 10 96-well Microtiter Plate Biofilm Cell Detachment Assay

The wells of a 96-well microtiter plate (Falcon no. 353072) were filledwith 100 μl of medium containing 10² to 10⁴ CFU of bacteria andincubated at 37° C. in 10% CO₂ for 20 hours. Ten μl of enzyme solution[1 mg ml⁻¹ in phosphate buffered saline (PBS)], or 10 μl of PBS in thecase of controls, was added to each well and the plates were incubatedfor an additional 6 hours. The wells were washed extensively underrunning tap water and the bacteria remaining attached to the surfacewere stained with crystal violet, rewashed, and destained with ethanolin accordance with procedures described by Kachlany et al. Mol.Microbiol. 2001 40:542-554). The optical density (O.D.) of theethanol-dye solution was measured in a BioRad Benchmark microtiter platereader set to 590 nm.

Example 11 Growth of Biofilms on Intravenous Catheters

Polyurethane catheters (1.1 mm diam, model 381434, Becton-Dickinson) andTeflon catheters (1.2 mm diam, model 3055, Critikon) were employed. Thetips of the catheters were plugged with sterile high vacuum grease toprevent media and dye from entering the lumen. Catheters were inoculatedand treated as described above for polystyrene rods. Precoating ofcatheters with dispersin B was carried out in PBS or in sodium phosphatebuffer (pH 9) for 10 minutes to 24 hours. In some cases, coatedcatheters were air dried for 24 hours before use. Teflon catheters werestained with crystal violet as previously described (Kaplan, J. B., andFine, D. H. Appl. Environ. Microbiol. 2002 68:4943-4950). Polyurethanecatheters were stained with 1% methylene blue in water for 2 minutes.

1. An isolated nucleic acid sequence encoding soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof whichpromotes detachment of bacterial or fungal cells from a biofilm.
 2. Theisolated nucleic acid sequence of claim 1 comprising a nucleic acidsequence with 50% sequence identity to at least 30 contiguousnucleotides of SEQ ID NO:1, 3, 5, 7 or
 9. 3. The isolated nucleic acidsequence of claim 1 comprising a nucleic acid sequence of SEQ ID NO:1,3, 5, 7 or
 9. 4. A nucleic acid sequence encoding a fusion polypeptidecomprising the isolated nucleic acid sequence of claim 1 and a secondnucleic acid sequence encoding a second polypeptide.
 5. A vectorcomprising the nucleic acid sequence of claim 1, 2, 3 or
 4. 6. A hostcell comprising the vector of claim
 5. 7. An isolated amino acidsequence encoded by the nucleic acid sequence of claim 1, 2, 3 or
 4. 8.An isolated soluble, β-N-acetylglucosaminidase protein or activefragment or variant thereof which promotes detachment of bacterial orfungal cells from a biofilm.
 9. The isolated soluble,β-N-acetylglucosaminidase protein or active fragment or variant thereofof claim 8 comprising SEQ ID NO:2, 4, 6, 8 or
 10. 10. A fusion proteincomprising the amino acid sequence of claim 8 or 9 and a secondpolypeptide.
 11. A pharmaceutical composition comprising an effectiveamount of the isolated soluble, β-N-acetylglucosaminidase protein oractive fragment or variant thereof of claim 8 or 9 and apharmaceutically acceptable carrier.
 12. A method for enhancing efficacyof an antibiotic against a bacterial infection comprising administeringthe pharmaceutical composition of claim 11 in combination with or priorto administration of the antibiotic.
 13. A medical device coated withthe isolated soluble, β-N-acetylglucosaminidase protein or activefragment or variant thereof of claim 8 or
 9. 14. A wound healing deviceimpregnated with the isolated soluble, β-N-acetylglucosaminidase proteinor active fragment or variant thereof of claim 8 or
 9. 15. A liquidantiseptic solution comprising the isolated soluble,β-N-acetylglucosaminidase protein or active fragment or variant thereofof claim 8 or
 9. 16. A method for inhibiting detachment of bacterial orfungal cells from biofilm comprising mutating a dspB gene of bacterialcells to inhibit detachment of bacterial or fungal cells from biofilms.17. A method for inhibiting detachment of bacterial or fungal cells frombiofilm comprising decreasing expression or levels of soluble,β-N-acetylglucosaminidase or inhibiting activity of soluble,β-N-acetylglucosaminidase in the bacterial cells so that detachment ofbacterial or fungal cells from the biofilm is decreased.
 18. An isolatedmutant of Actinobacillus actinomycetemcomitans which forms biofilmcolonies which tightly adhere to surfaces but which are unable torelease cells into the medium or spread over the surface.
 19. The mutantof claim 18 wherein the dspB gene is mutated.
 20. A method foridentifying an agent which modulates detachment of bacterial or fungalcells from biofilms comprising assessing an agent's ability to modulateactivity or expression or levels of soluble, β-N-acetylglucosaminidase.21. A method for promoting detachment of bacterial or fungal cells froma biofilm comprising contacting bacterial cells with soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof or anucleic acid sequence encoding soluble, β-N-acetylglucosaminidase or anactive fragment or variant thereof so that detachment of bacterial orfungal cells from a biofilm is promoted.
 22. A method for reducing riskof infection of an organism by bacteria or fungi on a medical device orsurgical instrument comprising contacting the medical device or surgicalinstrument with soluble, β-N-acetylglucosaminidase or an active fragmentor variant thereof prior to contacting the organism with the medicaldevice or surgical instrument.
 23. The method of claim 22 wherein themedical device is coated with the soluble, β-N-acetylglucosaminidase oran active fragment or variant thereof.
 24. The method of claim 23wherein the coating of soluble, β-N-acetylglucosaminidase or an activefragment or variant thereof is dried on the medical device.
 25. Themethod of claim 22 wherein the medical device is a catheter and thesoluble, β-N-acetylglucosaminidase or an active fragment or variantthereof is in a catheter lock solution in the catheter.
 26. A method forinhibiting, preventing or treating bacterial or fungal infectionscomprising administering to an organism soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof or anucleic acid sequence encoding soluble, β-N-acetylglucosaminidase or anactive fragment or variant thereof so that detachment of bacterial orfungal cells from a biofilm is promoted.
 27. The method of claim 26wherein the bacterial or fungal infection is from a bacterium or fungusthat produces a N-acetylglucosaminidase containing biofilmpolysaccharide that can be degraded by soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof. 28.The method of claim 26 wherein the soluble, β-N-acetylglucosaminidase oran active fragment or variant thereof or a nucleic acid sequenceencoding soluble, β-N-acetylglucosaminidase or an active fragment orvariant thereof is administered as a coating on a medical deviceimplanted in the organism.
 29. The method of claim 26 wherein thesoluble, β-N-acetylglucosaminidase or an active fragment or variantthereof or a nucleic acid sequence encoding soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof isadministered as a pharmaceutical composition.
 30. The method of claim 26wherein the soluble, β-N-acetylglucosaminidase or an active fragment orvariant thereof or a nucleic acid sequence encoding soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof isincorporated into a liquid disinfecting solution and applied topicallyto the subject prior to insertion of an implantable medical device. 31.The method of claim 26 wherein a wound dressing applied to the subjectis impregnated with the soluble, β-N-acetylglucosaminidase or an activefragment or variant thereof or a nucleic acid sequence encoding soluble,β-N-acetylglucosaminidase or an active fragment or variant thereof. 32.A primer pair which identifies bacteria with DspB homologs.
 33. Theprimer pair of claim 32 comprising SEQ ID NO:12 and SEQ ID NO:13.
 34. Akit for identifying bacteria with DspB homologs comprising the primerpair of claim 32 or 33 and instructions for use of the primer pair toidentify bacteria with DspB homologs.